Comparison of the reactivity of convalescent sera to different regions of Mycoplasma hyopneumoniae Mhp367 protein

Abstract:

[Background] Previous studies in our laboratory have demonstrated that Mycoplasma hyopneumoniae Mhp367 protein is a humoral immunodominant proteinic antigen. However, the reactivity of different regions of Mhp367 to the convalescent sera remains unclear. [Objective] The purpose of this study is to identify the reactivity of different regions of Mhp367 to the convalescent sera. [Methods] Nine DNA fragments of mhp367 were amplified with different primer combinations. The amplified products were respectively ligated into pGEX-6P-1, pGEX-4T-3, or pGEX-5X-3, and the ligation products were transformed into Escherichia coliDH5α competent cells. The constructed plasmids were extracted from the cells and confirmed by digestion with BamHⅠ and XhoⅠ and nucleotide sequencing. The identified plasmids were then transformed into E.coliBL21(DE3) to express target proteins (different fragments of Mhp367) under the induction of IPTG. After ultrasonic disruption of the recombinant bacteria, the protein was purified with glutathione-conjugated agarose beads. The expression of proteins was visualized by SDS-PAGE. ELISA was employed to identify the reactivity between different fragments of Mhp367 protein and convalescent sera. [Results] Nine recombinant strains that could express different fragments of Mhp367 in soluble form were successfully constructed. All of the nine fragments were humoral immunodominant. The region aa 394–524 in Mhp367 had the strongest reactivity with convalescent sera and was a good antigen fragment for vaccine candidates. [Conclusion] This study provides a candidate of genetic engineering subunit vaccines against M.hyopneumoniae.